A1 camera

SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in 30% sucrose until cryosections (7 μm) were cut (HM 560 Cryo-Star; Microm International GmbH, Walldorf, Germany) and subjected to routine haematoxylin and eosin staining [24 (link)].
For CD68 IHC, SIs were fixed in 4% neutral-buffered formalin for 24 h and then stored in PBS at 4 °C until paraffin embedding. Sections (7 mm) were deparaffinized, and staining was performed using a Leica Bond MAX immunostainer (Leica Microsystems, Wetzlar, Germany) and CD68 antibody (1:1,000; Cell Signaling Technologies, Danvers, MA). To visualize neutral lipids and nuclei, cryosections (7 mm) were stained with ORO (Sigma-Aldrich, St. Louis, MO) and DAPI (Akoya Bioscience, Marlborough, MA), respectively. Images were acquired at 60 × magnification (Apo 60 × Oil λS objective) using a Nikon Eclipse Ti microscope (NIKON Corporation, Tokyo, Japan) equipped with a Nikon A1 camera.

A minimum of 30 confocal images (60x objective; .5 μM slices) per condition were captured by a Nikon A1 camera (Nikon, Melville, NY, USA) linked to a Nikon Eclipse Ti2-E Inverted Microscope Imaging System (Nikon, Melville, NY, USA) for each channel (performed in triplicate, 2 independent experiments; N = 6 coverslips), which included RFP+ PDAC cells (red channel), FITC labeled dextran (green channel), and DAPI labeled nuclei (blue channel). First, each channel of stacked images was merged by max intensity using FIJI/Image J plugin “Z project”. Next, MetaMorph 7.8.1.0 offline software (Molecular Devices, Downingtown, PA) was used to quantify the amount of FITC-dextran taken up by RFP+ PDAC cells. RFP was used to generate a mask and the amount of FITC under that mask was queried (integrated density) and recorded. The integrated density of FITC under RFP was normalized to the DMSO treated CON PDAC cells (PDACc or PANC-1) for each experiment.